αvβ3 integrin serves as a receptor for a variety of extracellular matrix proteins displaying the arginine-glycine-aspartic acid (RGD) tripeptide sequence. These proteins include vitronectin, fibronectin, fibrinogen, laminin, collagen, Von Willibrand's factor, osteoponin, and adenovirus particles (Jin, H. and J. Varner, Br. J. Cancer, 2004. 90(3): p. 561-5). αvβ3 integrin, expressed on the surface of various normal and cancer cell types, is involved in multiple physiological processes including angiogenesis, apoptosis, and bone resorption. During angiogenesis, attachment of endothelial αvβ3 integrin to the extracellular matrix is required for the survival and maturation of newly forming blood vessels and ligands against αvβ3 integrin induce apoptosis of angiogenic vascular cells, leaving pre-existing quiescent blood vessels unaffected. In addition, αvβ3 integrin has been observed to be over-expressed on metastatic tumor cells such as malignant melanoma and glioblastoma. Since integrin plays a key role in angiogenesis and metastasis of human tumors, αvβ3 integrin ligands are of great interest to advances in targeted-therapy and cancer imaging.
RGD motif was described in early 1980's by several groups such as Michael D. Pierschbacher and Erkki Ruoslahti (Pierschbacher, M. D. and E. Ruoslahti, Nature, 1984. 309(5963): p. 30-3). Since then, many RGD analogues have been designed and synthesized (Haubner R., G. R., Diefenbach B., Goodman S. L., Jonczyk A., Kessler H., J. Am. Chem. Soc., 1996. 118(32): p. 13). Many naturally occurring snake venoms also contain the RGD motif (Markland, F. S., Toxicon, 1998. 36(12): p. 1749-8000). Phage-display peptide library screening has been used to identify peptide ligands targeting various integrins, thereby elucidating unique integrin or tissue targeting peptides. Using this screening methodology, the nonapeptide, CDCRGDCFC (SEQ ID NO:1), was identified to be highly selective against αv integrins (Koivunen, E., B. Wang, and E. Ruoslahti, Biotechnology (N Y), 1995. 13(3): p. 265-70). Another “design approach” based on “spatial screening” of cyclopeptides, in which conformational restriction is induced by variation of the ring size, amino acid chirality and retro-inverso structures, N-methylation of peptide backbone, or introduction of constraining structural elements, have led to the discovery of Cilengitide (Haubner R., G. R., Diefenbach B., Goodman S. L., Jonczyk A., Kessler H., J. Am. Chem. Soc., 1996. 118(32): p. 13 and Dechantsreiter, M. A., et al.,. J Med Chem, 1999. 42(16): p. 3033-40). This cyclic peptide, cyclo(RGD-(NMe)V-) (SEQ ID NO:2), binds strongly and relatively selectively to αvβ3 integrin, and is now in clinical trials for the treatment of several different kinds of cancers (Friess, H., et al., BMC Cancer, 2006. 6: p. 285.; Hariharan, S., et al., Ann Oncol, 2007. 18(8): p. 1400-7; MacDonald, T. J., et al., J. Clin. Oncol., 2008. 26(6): p. 919-24.; and Nabors, L. B., et al., J. Clin. Oncol., 2007. 25(13): p. 1651-7). Some other cyclopeptides and their derivatives which bind integrin are disclosed in U.S. Pat. No. 6,610,826. However, due to intrinsic constraints of these screening processes, only L-amino acid residues can be included in the phage display peptide libraries and the number of peptides that can be simultaneously tested in the “spatial screening” process is rather limited.
There is a need for better and different binding agents of αvβ3 integrin that can have improved targeting efficacy, lower nonspecific binding to normal organs and carry payloads of therapeutics and/or diagnostics. Surprisingly, the present invention satisfies this and other needs.